Int J Hematol (2008) 88:374–380 DOI 10.1007/s12185-008-0165-5
ORIGINAL ARTICLE
Prognostic relevance of serum levels and cellular expression of adiponectin in B-cell chronic lymphocytic leukemia Stefano Molica Æ Gaetano Vitelli Æ Giovanna Cutrona Æ Katia Todoerti Æ Rosanna Mirabelli Æ Giovanna Digiesi Æ Diana Giannarelli Æ Isabella Sperduti Æ Matteo Molica Æ Massimo Gentile Æ Fortunato Morabito Æ Antonino Neri Æ Manlio Ferrarini
Received: 18 March 2008 / Revised: 18 July 2008 / Accepted: 20 August 2008 / Published online: 27 September 2008 Ó The Japanese Society of Hematology 2008
Abstract The correlation between well-established biological parameters of prognostic relevance in B-cell chronic lymphocytic leukaemia (CLL) [i.e., mutational status of the immunoglobulin heavy chain variable region (IgVH), ZAP-70- and CD38-expression] and adiponectin serum concentration was evaluated in a cohort of 69 previously untreated Binet stage A CLL patients. Adiponectin levels inversely correlated with absolute peripheral blood lymphocyte count (r = -0.254; P = 0.03), CD38-positive CLL cells (r = -0.294; P = 0.04) and ZAP-70 (r = -0.285; P = 0.03). The univariate Cox proportional hazard model demonstrated that, in addition with lower serum levels of adiponectin (P = 0.01), the unmutated IgVH condition (P = 0.002) and ZAP-70-positivity (P = 0.02) were associated with a shorter time to first treatment (TFT). However, in multivariate analysis only S. Molica (&) R. Mirabelli Medical Oncology Unit, Hematology-Oncology Department, Azienda Ospedaliera Pugliese-Ciaccio, Viale Pio X, 88100 Catanzaro, Italy e-mail:
[email protected] G. Vitelli G. Digiesi D. Giannarelli I. Sperduti Clinical Pathology, IRCCS Regina Elena, Rome, Italy G. Cutrona M. Ferrarini Medical Oncology C, IST, Genoa, Italy M. Gentile F. Morabito Hematology Department, Az. Ospedaliera, Cosenza, Italy K. Todoerti A. Neri Leukemia Study Center, Department of Medical Sciences, University Milano, Hematology 1, Fondazione IRCCS Policlinico, Milan, Italy M. Molica Medical School La Sapienza, Rome, Italy
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ZAP-70 positivity emerged as predictor of the TFT (P = 0.008). The levels of adiponectin in CLL were evaluated in 60 patients from an independent cohort investigated by gene expression profiling. Adiponectin gene expression was invariably low suggesting a limited (if any) role of leukemic cells in the production of circulating adiponectin levels. In contrast, both adiponectin receptor 1 (AdipoR1) and AdipoR2 mRNA were highly expressed by CLL cells with a degree of inter-patient variability. Our results, although preliminary, lend support to the idea that adiponectin secretion by bone marrow adipocytes might represent a possible promising drug target in the field of hematology. Keywords Early CLL Prognosis Adiponectin IgVH mutations ZAP-70 CD38
1 Introduction One of the genes most closely related to IgVH mutation status is lipoprotein lipase (LPL). High expression of this central enzyme of lipid metabolism was associated with unmutated B-CLL in almost all profiling studies, regardless of methodology or B-cell selection process [1, 2]. Lipoprotein lipase is normally produced and secreted by adipocytes, macrophages, cardiac, and skeletal muscle cells and catalyzes the hydrolysis of the triacylglycerol component of chylomicrons and very low-density lipoproteins (VLDL) [3]. Interestingly, adipocytes, the most abundant stromal cell in human adult bone marrow (BM), produce in significant amounts adiponectin, and express both adiponectin receptor subtypes adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) [4]. Adiponectin along with leptin are among the most probable and putative adipocytokines
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to control hematopoiesis. As matter of fact, adiponectin serum levels were reported to be inversely correlated with red blood cell counts, white blood cell counts, and platelet counts in peripheral blood [5]. Adiponectin serum level has been inversely associated with childhood acute myeloblastic leukemia (AML), and not with other types of childhood leukemia [6]. In both chronic myeloproliferative and lymphoproliferative disorders circulating levels of adiponectin were lower in comparison to age, sex, and body mass index matched control; although treatment with interferon led to a significant increase of adiponectin plasma levels in patients with chronic myeloproliferative disorders [7]. With this in mind we assessed serum levels of adiponectin in 69 patients with Binet stage A CLL and these values were retrospectively correlated with Rai stage, b2microglobulin (b2-m), mutational status of the IgVH, ZAP70- and CD38-expression and clinical outcome. Finally, looking for cellular source of adiponectin we investigated the presence of adiponectin and its receptors in CLL cells at gene expression level in 60 B-CLL patients belonging to an independent series.
2 Materials and methods
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biopsies were performed in 45 patients (67.1%) and the pattern of BM involvement was evaluated according to Rozman et al. [10]. Lymphocyte doubling time (LDT) was available in 56 patients (83.5%) followed-up for more than 12 months and assessed according to Montserrat et al. [11] criteria. 2.3 Measurement of adiponectin and other cytokines Serum adiponectin concentrations were measured in duplicate with a quantitative sandwich enzyme immunoassay (ELISA) technique (Human Adiponectin Quantikine Colorimetric ELISA, R & D Systems, Minneapolis, MN, USA). Calibrations were carried out with human adiponectin standards. Optical densities at 450 nm were measured with a microtiter plate reader. A standard curve was created by plotting the logarithm of the mean absorbance of each standard versus the logarithm of the cytokine concentration. Concentrations were expressed in lg/mL and the coefficient of variation (CV) reported by the manufacturer for inter-assay and intra-assay determinations ranged from 6.9 to 8.8% and from 2.5 to 4.7%, respectively. Serum vascular endothelial growth factor (VEGF) levels were measured with a quantitative ELISA (Human VEGF, QuantikineÒ; R & D Systems, Minneapolis, MN, USA).
2.1 CLL patients’ cohorts 2.4 Detection of ZAP-70 Two independent series of previously untreated Binet stage A CLL patients were investigated. The first series included 69 patients followed at the Medical Oncology Department of Catanzaro; the second cohort included 60 Binet stage A CLL patients from a cooperative database specifically devised to investigate the gene expression profiling of CLL cells. Both patients cohorts also were investigated for other prognostic factors such as IgVH mutational status, and ZAP-70 and CD38 expression (see below). 2.2 Characteristics of patients evaluated for serum adiponectin level Sixty-nine patients were selected based on the availability of frozen serum samples. Their median age was 64 years (range 40–83) and the male to female ratio 41:28. Routine laboratory studies consisted of complete blood count with differential, platelet count, and blood chemistry including b2-m and LDH, as well as immunophenotyping to establish the diagnosis of typical CLL [8]. Physical examination, chest X-ray, and abdominal ultrasound were performed in all instances. Patients were also clinically staged according to the Rai staging system [9] and distributed as follows: stage 0, 48 (68.6%) and stage I, 15 (10.4%) and stage II (20.8%). BM
Mononuclear cells (MNC) from CLL cases were isolated by Ficoll-Hypaque (Seromed, Biochrom KG, Berlin, Germany) density gradient centrifugation, stained with CD19-PE and CD3 PE-CY7 (Becton Dickinson, San Diego, CA), fixed and permeabilized with fix and perm reagents (Caltag Laboratories, Burlingame, CA) at the concentration of 5 9 105 cells/50 ll. The cells were subsequently exposed to ZAP-70 mAb conjugated with FITC (clone 2F3.2, Upstate, lake Placid, NY) and analyzed by flow cytometry (FacsCalibur Becton Dickinson). A CD33 FITC (Becton Dikinson) mAb of the same IgG1 subclass was used as negative control of ZAP-70 [12]. In 60 B-cell CLL patients analyzed for gene expression array determination of ZAP-70 was carried out by Western blot as described [13, 14]. Briefly, constant amounts of protein from cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% acrylamide (SDS-PAGE), transferred to polyvinylidenefluoride (PVDF) Hybond-P membranes (Amersham PharmaciaBiotech, Buckinghamshire, UK), and exposed to primary mouse mAb specific ZAP-70, (clone 2F3.2 Upstate, Lake Placid, NY) or actin (sigma Aldrich, Milan, Italy) and then horseradish peroxidase (HRP)-conjugated Goat anti-mouse IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, USA).
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Peroxidase activity was revealed by chemiluminescence. H9 T cells expressing high levels of ZAP-70 were used as positive controls. ZAP-70 positivity was expressed as percentage of ZAP-70 positive cells, determined by comparing the intensity of the ZAP-70 band observed for a given CLL to that of control suspension containing known proportions of T cells. 2.5 CD-38 evaluation Direct immunofluorescent staining was performed with CD19 FITC/PE, CD23 PE, CD38 PE, and Cy-Chrome (Becton Dickinson & Co., Sunnyvale, CA). CD19 FITC, CD23 PE, and CD5 Cy-Chrome were used to assess the proportion of CLL cells in the suspensions; the proportion of CD38-positive leukemic cells was determined by triple staining for CD19 FITC, CD38 PE, and CD5 Cy-Chrome. The cells were analyzed using a FACS-sort flow cytometer (Becton Dickinson & Co). 2.6 IgVH gene sequencing The mutational status of the IgVH genes was determined as previously described [13]. Sequences with C2% difference in IgVH from the most similar germline gene were considered mutated (‘‘mutated B-CLL’’). Sequences with \2% difference were considered unmutated (‘‘unmutated B-CLL’’). 2.7 Gene expression T cells, NK cells and monocytes were removed from PBMNC of CLL patients by CD3, CD56, CD16, and CD14 monoclonal antibody (mAb) treatment (Becton Dickinson, San Diego, CA) followed by magnetic beads (Goat Anti-Mouse IgG D ynabeads, Dynal Biotech ASA, Oslo, Norway). The purity of the B cells was assessed by flow cytometry by the determination of the proportions of CD5/CD19/CD23 triple positive B cells in the suspension. Purified normal peripheral blood B cells were obtained from six normal donors. Total RNA was isolated using the TRIzol Reagent (Life Technologies, Inc., Rockville, MD, USA) and then purified using the RNeasyÒ total RNA Isolation Kit (Qiagen, Valencia, CA). Preparation of biotin-labeled cRNA, hybridization to GeneChipÒ Human Genome U133A Arrays (Affymetrix Inc., Santa Clara, CA) and scanning of the chips (7G Scanner, Affymetrix Inc., Santa Clara, CA) were carried out according to manufacturer’s protocols. The images were analysed using AffymetrixÒ GeneChip Operating Software (GCOS) 1.4 and the probe level data were converted to expression values using the bioconductor function for Robust multiarray average (RMA) procedure, in which perfect match
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values are background adjusted and normalized using quantile–quantile normalization, as previously described [15]. Expression data of the 60 B-CLLs proprietary database were deposited in the National Centre for Biotechnology Information’s Gene Expression Omnibus (GEO; http://www.ncbi.nlm.mih.gov/geo) and are available through GEO series accession number GSE11038. 2.8 Clinico-hematological studies and evaluation of time-to-first treatment Serum levels of adiponectin were correlated with main clinico-hematological variables, such as Rai substages, absolute PB lymphocytosis, LDH, b2-m, IgVH mutational status, CD38 and ZAP-70 expression. Correlation with serum levels of VEGF was also assessed. Furthermore, serum adiponectin was investigated as a predictor of clinical outcome by using as end-point the time to first treatment (TFT), a reliable parameter for assessing disease-progression (DP). As described previously [16], DP has an important impact on overall survival of CLL patients in early stage and hence it can surrogate overall survival, thus shortening the duration of clinical studies in CLL. 2.9 Statistics Spearman correlations, Mann-Whitney test and the corrected v2 test were applied to compare groups. Progression-free survival (PFS) curves were plotted according to Kaplan– Meier method and compared with the log-rank test. To screen for patients who likely could experience a need for chemotherapy, cutoffs of adiponectin level corresponding to the highest Youden index were chosen. The index combines information on sensitivity and specificity, giving equal weight to each, and measures the percentage gain in certainty of predicting the TFT. If adiponectin level at a definite cutoff has an index of 0, it has no predictive power; if the cutoff has an index of 100% TFT is perfectly predicted. Adiponectin level cutoffs set at 25th, 50th and 75th percentiles gave an index of 12, 48, and 24, respectively. Thus, the median value of adiponectin level was chosen as cutoff. The hazard risk and the confidence limits were estimated for each variable using the Cox univariate model and adopting the most suitable prognostic category as referent group. A multivariate Cox proportional hazard model was also developed using stepwise regression (forward selection) with predictive variables which were significant in the univariate analyses. Enter limit and remove limit were P = 0.10 and P = 0.15, respectively. The SPSS (11.0) statistical program was used for analysis.
Adiponectin in CLL
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Fig. 1 Correlation between adiponectin serum levels and percentage of CD38-positive cells (a), or ZAP-70-positive cells (b). Either CD38 or ZAP70 were evaluated in flow cytometry
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r = - 0.294; P = 0.02
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3.1 Serum levels of adiponectin in CLL patients and relationship with other markers Adiponectin serum levels in CLL patients varied between 1.1 and 22.0 lg/mL, with a median of 5.88 lg/mL. In healthy age- and sex-matched controls, the median was 5.4 lg/mL, with a range from 1.2 to 11.8 lg/mL. Overall, the levels of adiponectin in patients with CLL were no significantly different from those of healthy controls (Mann-Whitney test, P = 0.6583). Interestingly, either all normal controls or CLL patients had a body mass index (BMI)\30 kg/m2. Adiponectin levels did not correlate with patients’ age (P = 0.167), b2-m (P = 0.947), LDH (P = 0.803), hemoglobin (P = 0.761), platelet count (P = 0.517), Rai substages (P = 0.143) and IgVH mutational status (P = 0.817). In contrast, there was a significant inverse association between adiponectin and absolute peripheral blood lymphocyte count (r = -0.254; P = 0.03), and CD38 (r = -0.294; P = 0.04) (Fig. 1a) and ZAP-70 (r = -0.285; P = 0.03) (Fig. 1b), and a direct correlation with circulating levels of VEGF (r = 0.337; P = 0.006). Finally, higher circulating levels of adiponectin were more frequently found in women than in men (P = 0.0002). 3.2 The impact of serum adiponectin levels on TFT The risk of DP according to known and putative prognostic parameters was evaluated in our cohort of Binet stage A patients as TFT. The univariate Cox proportional hazard model demonstrated that in addition with lower serum levels of adiponectin [hazard ratio (HR) 2.936; 95% confidence interval (CI) 1.245–6.919; P = 0.01], the unmutated IgVH condition (HR 6.378; 95% CI 1.971– 20.642; P = 0.002) and ZAP-70-positivity (HR 3.314; 95% CI 1.128–9.736; P = 0.02) were associated with a shorter TFT. As shown in Fig. 2, Kaplan–Meier estimate of patients TFT, plotted after setting the best cut-off for adiponectin (i.e., 5.88 lg/mL), demonstrated that low adiponectin concentration was associated with a shorter
Percentage of patients without therapy
3 Results Serum adiponectin > median 0.8 0.6
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Fig. 2 Kaplan-Meier curves of time to first treatment of Binet stage A patients stratified on the basis of median value of circulating adiponectin level (i.e., 5.88 lg/mL)
TFT. As a matter of fact patients with serum adiponectin lower than 5.88 pg/mL had a median TFT of 36 months while median time of TFT was not reached by patients with adiponectin levels higher than 5.88 lg/mL (P = 0.01). Finally, in multivariate analysis only ZAP-70 positivity emerged as predictor of the TFT (HR 5.187; 95% CI 1.539–17.489; P = 0.008). 3.3 Evaluation of adiponectin and adioponectin receptors gene transcript In this study, we examined the presence of adiponectin and AdipoR1 and AdipoR2 receptors in B-CLL cells at gene expression level. To this purpose gene expression of purified cells from 60 B-CLL patients and six peripheral blood B cells from healthy donors were profiled using highdensity oligonucleotide microarrays. As evidenced in Fig. 3a, the RMA intensity values for adiponectin gene transcripts denote a homogeneous low expression in BCLL cells as in healthy donors samples. In contrast, both AdipoR1 and AdipoR2 mRNA receptors were highly expressed with a degree of inter-patient variability, showing also an average fold change of 1.7 in B-CLL patients versus normal samples expression (Fig. 3b, c). Finally, we looked at correlation between RMA intensity values of adiponectin and LPL gene transcripts.
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a ADIPOQ
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0
Fig. 3 Absolute RMA intensity values for adiponectin (ADIPOQ) (a), adiponectin receptor1 (ADIPOR1) (b), and adiponectin receptor2 (ADIPOR2) (c), transcripts in 60 B-CLL patients and six peripheral blood B cells (N-01–N-06) dataset, assessed by microarray analysis. Samples were ordered on the base of the expression values of each
specific probeset in healthy donors (stripped bars) and B-CLLs groups (white bars), respectively. Since B-CLL samples were collected in different Italian Institutions, they were named accordingly to their provenience (CS Cosenza, GE Genova, RC Reggio Calabria, TS Trieste)
Interestingly, an inverse correlation between gene expression level of LPL and adiponectin was found (r = -0.227; P = 0.04).
several observations link adiponectin with mechanisms of cancer development and progression [17–21]. Adiponectin suppresses the expression of adhesion molecules in vascular endothelial cells and cytokine production from monocytes/macrophages, thus inhibiting the inflammatory processes [22]. In addition, adiponectin is an important negative regulator of immune systems, hematopoiesis and suppresses colony formation from colony forming unit granulocyte and macrophage [23].
4 Discussion The association between circulating levels of adiponectin and clinical outcome has been recently evaluated and
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Our aim was also to evaluate the relationship between adiponectin serum levels, newer biological variables and conventional clinico-hematological parameters of prognostic significance, by testing their impact on the need for therapy. The results of the present study clearly show that lower circulating levels of adiponectin were more frequently found among early CLL patients with an aggressive phenotype as defined by higher expression of ZAP-70 or CD38. In keeping with this finding, lower circulating levels of adiponectin identified a subset of Binet stage A CLL patients with shorter TFT. However, multivariate Cox analysis demonstrated that only ZAP-70 expression retained its prognostic significance therefore leading to conclude that adiponectin may not replace the need for the determination of ZAP-70 and IgVH mutational status [24]. Looking for cellular source of adiponectin we investigated the presence of adiponectin in B-CLL cells at gene expression level in 60 B-CLL patients belonging to an independent series. We provide evidence for the presence of a low adiponectin gene expression transcript in B-CLL cells. This observation is in keeping with recently published data from Iversen et al. [12] who evaluated adiponectin gene expression in BM AML blasts. Adiponectin transcripts were not found in AML blast cells thus suggesting that adiponectin production relies on adipocytes present in the BM fat [12]. Recent results suggest an association of decreased LPL activity with low plasma adiponectin that is independent of systemic inflammation and insulin resistance [25]. Interestingly, in our series of CLL patients investigated by gene expression profiling we found that adiponectin transcript, although expressed at low level, inversely correlated with LPL gene expression transcript, one of the genes most closely related to IgVH mutation status as well as with prognosis. Finally, we observed that AdipoR1 and AdipoR2 receptors were highly expressed in B-cell CLL patients. Arditi et al. [26] found that higher adiponectin concentrations inhibit proliferation but causes no apoptosis of MCF7 breast cancer cells. Also in CLL the effect of adiponectin on cell proliferation might be most likely specific and adiponectin receptor-mediated, therefore the association between low serum adiponectin levels and increased risk of DP in patients with early CLL might be explained, at least in part, by the direct effect of adiponectin on the leukemic cells. In conclusion, our results, although preliminary, lend support to the idea that adiponectin secretion by BM adipocytes might represent a possible promising drug target in the field of hematology. Recombinant adypocytokines might represent future drugs in hematology for lineage-
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specific stimulation or inhibition of cell growth, differentiation and proliferation.
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